克隆山羊成纤维细胞系的建立与生物学特性的研究

Establishment and Biological Characteristics of Fibroblast Cells Derived from Somatic Cloned Goat

为研究克隆黄淮白山羊耳部皮肤成纤维细胞(cGSF)的体外培养及其生物学特性。通过组织块接种培养法和胰蛋白酶消化法均可以获得原代cGSF,但在接种培养24 h后,组织块法收获的细胞贴壁率和细胞量要明显高于胰酶消化法。就生长特征而言,cGSF的生长曲线也呈现典型"S"型,到达指数增长期、平台期的时间分别为第3,6天,早于普通黄淮白山羊的皮肤成纤维细胞(GSF),后者分别为第4,8天。cGSF原代(P0)、第2代(P2)、第8代(P8)细胞的染色体数目异常率均高于相同代数的GSF。在相同转染条件下,cGSF在转染p EGFP-N1后,再经G418筛选亦可形成克隆点,扩增获得阳性细胞系。总之,源自克隆山羊的体细胞体外生长、转基因后的生物学特征有别于普通动物分离得到的细胞系。因此,利用来自克隆动物的体细胞开展研究,选择分离培养、纯化、转基因方法时需要适当调整。

The present study aimed to establish ear skin fibroblasts（ c GSF） from a cloned Huanghuai white goat and explore the biological characteristics of the established c GSF cell line. The results were as follows： Both explants adherence culture（ A method） and trypsin digestion（ B method） could result in the successful growth of primary fibroblasts. However,after 24 h in culture,adherence rate of cells harvested from A method was higher than that of B method. The amount of harvested cells using A method was also greater than using B method. As for the growing characteristics,the growth curve of c GSF cells was typical ＂ S＂ type. For the time spent on reaching to the exponential growth phase（ Day 3 Vs. Day 4） and the platform phase（ Day 6 Vs. Day 8）,c GSF was earlier than control（ noncloned goat fibroblast cells）. We also found that rate of chromosomal abnormalities in cells from cloned goats was higher than those from non-cloned. In order to test feasibility of transfection using cGSF,pEGFP-N1 plasmid was subjected to be transfected with cultured c GSF,and we found that c GSF cells were more easier to form clonies after G418 screen. Therefore,if cells from cloned animals are employed in transgenic research,appropriate readjustment in cell isolation,in vitro culture,transfection and positive cell screen and enrichment should be done accordingly. In all,cells in vitro derived form cloned goat show differences in biological characteristics from cell lines of non-cloned goat.